About the Disease and Pulmonary Surfactant Protein A
Tuberculosis (TB) is caused by Mycobacterium tuberculosis [Grange J M and Bishop P J. A tribute to Robert Kosh's discovery of the tubercle bacillus. ‘UberTuberkulosis’ 1982; 62:1-17]. M. bovis and M. africanum which are closely related organisms, can also infect human beings [Young L S. Mycobacterial diseases and the compromised host. Clin Infect Dis 1993; 17:8436-8441]. In addition, there are other human pathogens belonging to the genus Mycobacterium which are related to M. tuberculosis, such as M. avium and M. leprae, that represent important pathogens.
There is a growing concern about the worldwide increasing incidence of TB, a leading killer disease [Raviglione M C, Snider D E Jr, Kochi A. Global epidemiology of tuberculosis. Morbidity and mortality of a worldwide epidemic. JAMA 1995; 273:220-6]. It kills three million people all over the world annually. One third of the world population is estimated to be infected with M. tuberculosis asymptomatically, and in the year 1995, more people died of TB than in any earlier year in the history of mankind [Kochi A. WHO report on tuberculosis epidemic. 1996].
According to a recent report by WHO on tuberculosis epidemic (WHO Annual Report, 2000), it is estimated that between 2000 and 2020, nearly one billion people will carry the tuberculosis bacteria, 200 million people waaill get sick, and 35 million will die of TB, if control and preventive measures are not strengthened.
Tuberculosis primarily affects lungs (Pulmonary tuberculosis), although in one-third of the cases other organs are also affected. It is mainly transmitted by patients with infectious pulmonary tuberculosis in the form of airborne droplet nuclei produced during coughing and sneezing [Riley R L and Grady F O. Airborne infection. Transmission and control. The Macmillan Co., New York. 1961]. The disease, if untreated, can be fatal within a few years.
Mycobacterium tuberculosis is a facultative intracellular pathogen of alveolar macrophages located in the apical region of lung [Balasubramanian V, Weigeshaus E H, Taylor B T, Smith D W. Pathogenesis of tuberculosis: pathway to apical localization. Tuber Lung Dis 1994; 75:168-78]. It employs several mechanisms to enter human macrophages, where it survives well and from where it continues its pathogenic life cycle. Innate immune system is an integral part of host defense against tuberculosis [Schluger N W, Rom W N. The host immune response to tuberculosis. Am J Respir Crit Care Med 1998; 157:679-91]. Pulmonary surfactant, especially surfactant proteins A and D, has evolved as an important component involved in innate immunity as well as regulation of inflammatory processes of the lung [Mason R J, Greene K, Voelker D R. Surfactant protein A and surfactant protein D in health and disease. Am J Physiol 1998; 275:L1-13].
Human lung surfactant protein A (SP-A) binds M. tuberculosis and enhances attachment of M. tuberculosis to alveolar macrophages [Gaynor C D, McCormack F X, Voelker D R, Me Gowan S E, Schlesinger L S. Pulmonary surfactant protein A mediates enhanced phagocytosis of Mycobacterium tuberculosis by a direct interaction with human macrophages. J Immunol 1995; 155:5343-51.; Pasula R, Downing J F, Wright J R, Kachel D L, Davis T E Jr, Martin W J 2IX-Surfactant protein A (SP-A) mediates attachment of Mycobacterium tuberculosis to murine alveolar macrophages. Am J Respir Cell Mol Biol 1997; 17:209-17]. SP-A has also been implicated as an opsonin enhancing phagocytosis of mycobacteria by alveolar macrophages [Weikert L F, Edwards K, Chroneos Z C, Hager C, Hoffman L, Shepherd V L. SP-A enhances uptake of bacillus Calmette-Guerin by macrophages through a specific SP-A receptor. Am J Physiol 1997; 272:L989-95].
In view of the importance of SP-A in host defense against tuberculosis, structural and functional changes in SP-A may affect the outcome of host-pathogen interaction.
SP-A is the major non-serum pulmonary surfactant-associated protein with 18 functional units showing significant structural similarity to Clq [Crouch E C. Collectins and pulmonary host defense Am J Respir Cell Mol Biol 1998 19:177-201]. The human genome contains two highly similar SP-A genes (SP-A1 and SP-A2) and a pseudogene that corresponds to 3′ half of the SP-A1 gene [Hoover R R, Floras J. Organization of the human SP-A and SP-D loci at 10q22-q23. Physical and radiation hybrid mapping reveal gene order and orientation. Am J Respir Cell Mol Biol 1998; 18:353-62].
In adult human lung tissue, the ratio of SP-A2 to SP-A1 mRNA transcripts has been observed to be 3:1 [McCormick S M, Boggaram V, Mendelson C R. Characterization of mRNA transcripts and organization of human SP-A1 and SP-A2 genes. Am J Physiol 1994; 266:L354-66]. Contributions of SP-A 1 and SP-A2 genes to the SP-A transcript vary at different gestation times and SP-A2 gene is more responsive to glucocorticoids [Kumar A R, Snyder J M. Differential regulation of SP-A1 and SP-A2 genes by cAMP, glucocorticoids, and insulin. Am J Physiol 1998; 274:L177-85].
For each human SP-A gene, based on sequence differences within the coding region more than 30 genetic variants (alleles) have been reported [DiAngelo S, Lin Z, Wang G, Phillips S, Ramet M, Luo J, Floras J. Novel, non-radioactive, simple and multiplex PCR-cRFLP methods for genotyping human SP-A and SP-D marker alleles. Dis Markers 1999; 15:269-81]. Some of the allelic variants of SP-A1 and SP-A2 have been associated with respiratory diseases such as Respiratory distress syndrome (RDS) and Chronic obstructive pulmonary disease (COPD) [Karinch A M, deMello D E, Floros J. Effect of genotype on the levels of surfactant protein A mRNA and on the SP-A2 splice variants in adult humans. Biochem J 1997; 321:39-47; Kala P, Ten Have T, Nielsen H, Dunn M, Floros J. Association of pulmonary surfactant protein A (SP-A) gene and respiratory distress syndrome: interaction with SP-B. Pediatr Res 1998; 43:169-77; Ramet M, Haataja R, Marttila R, Hamalainen A M, Knip M, Hallman M. Human surfactant Protein˜A gene locus for genetic studies in the Finnish population. Dis Markers 2000; 16:119-24; Guo X, Lin H M, Lin Z, Montano M, Sansores R, Wang G, DiAngelo S, Pardo A, Selman M, Floros J. Polymorphisms of surfactant protein gene A, B, D, and of SP-B-linked microsatellite markers in COPD of a Mexican population. Chest 2000; 117:2498-508].
A recent case-control association study showed that the frequency of certain alleles of SP-A is increased in individuals with tuberculosis in Mexican population [Floros J, Lin H M, Garcia A, Salazar M A, Guo X, DiAngelo S, Montano M, Luo J, Pardo A, Selman M. Surfactant protein genetic marker alleles identify a subgroup of tuberculosis in a Mexican population. J Infect Dis 2000; 182:1473-8]. However, the allelic variants of SP-A1 and SP-A2 have not been identified so far in developing nations like India where pulmonary tuberculosis is highly prevalent. Collagen region of SP-A has been shown to be involved in receptor binding on macrophages, regulation of surfactant secretion and lipid uptake by type II cells [McCormack F X, Damodarasamy M, Elhalwagi B M. Deletion mapping of N-terminal domains of surfactant protein A. The N-terminal segment is required for phospholipid aggregation and specific inhibition of surfactant secretion. J Biol Chem 1999; 274:3173-81]. Two of the SNPs (1649 C/G and 1660 A/G) in the collagen region have shown association with patients of allergic bronchopulmonary aspergillosis [U.S. patent application Ser. No. 10/102,731].
Disease Loci Identified Till Now and Their Associations
Association studies involve typing a genetic polymorphism in a large number of unrelated individuals with the disease of interest and a group of healthy ethnically matched controls. Genes which could be involved in the development of tuberculosis are HLA, Nrampl, Tumor Necrosis Factor, mannose binding protein, Vitamin D receptor, Interferon-y receptor, Interleukin 10, Interleukin 1α, 1β and RA, Complement receptor 1, 1C AMI, Fucosyltransferase 2, Inducible nitric oxide synthase, Chemokine receptors, Interleukin 4, Tfy cluster and Interleukin 6 etc. All these candidate gene contain known polymorphisms but there may be many other genes which could play a role in tuberculosis susceptibility and are worthy of investigation [Richard Bellamy. Genetic susceptibility to tuberculosis in human populations. Thorax 1998; 53: 588-593.].
Attempts to identify the actual gene involved in host susceptibility to tuberculosis have recently focussed on the human leucocyte antigen (HLA) systems. Associations have been found between the class I HLA antigens A10 and B8 and with the class II antigens DR2 [Brahmajothi V, Pitchappan R M, Kakkanaiah V N, et al The association of tuberculosis and HLA in South India. Tubercle 1991; 72: 123-32; Singh S P N, Mehra N K, Dingley H B, et al Human leucosyte antigen (HLA)-linked control of susceptibility to tuberculosis and association with HLA-DR types. J Infect Dis 1983; 148:676-81]. However, these associations have not been consistently demonstrated [Cox R A, Down M, Neimes R E, et al Immunogenetic analysis of human tuberculosis. J Infect Dis 1988; 158: 1302-8] and could account for only a small part of the significant genetic component in tuberculosis susceptibility identified by the twin study.
Frequency comparisons of surfactant protein marker alleles in tuberculosis patients and healthy controls subjects have also been performed. Regression analysis of the tuberculosis and the tuberculin-skin test positive groups revealed, on the basis of odds ration, tuberculosis susceptibility (DA11_C and GATA 3) and protective (AAGG—2) marker alleles. Similarly, 1 A3,6A4, and B1013_A and protective AAGG—2, and AAGG—7 marker alleles were observed. Moreover, interactions were seen between alleles 6A2 and 1 A3 and between 1A3 and B1013_A. The findings indicate a possible involvement of SP alleles in tuberculosis pathogenesis [Joanna Floros, Hung-Mo Lin, Andrea Garcia, Miguel Angel Salazar, Xiaoxuan Guo, Susan DiAngelo, Martha Montano, Junming Luo, Annie Pardo, and Moises Selman. Surfactant Protein Genetic Marker Alleles Identify a Subgroup of Tuberculosis in a Mexican Population. J Infect. Dis. 2000; 182: 1473-8].
Single Nucleotide Polymorphisms
SNP's occur with greater frequency and are spaced more uniformly throughout the genome than other forms of polymorphism. The greater frequency and uniformity of SNP's means that there is a greater probability that such a polymorphism will be found in close proximity to a genetic locus of interest than would be the case for other polymorphisms. Also, the different forms of characterised SNP's are often easier to distinguish than other types of polymorphisms (eg. by use of assays employing allele-specific hybridisation probes or primers).
The Applicant after much research and study has demonstrated the application of SNP's (G1649C and A1660G) in human SP-A2 gene for use as molecular diagnosis and prediction of an individual's disease susceptibility to pulmonary tuberculosis, and/or the genetic analysis of SP-A2 gene in Indian population. The novelty of the present invention is in providing a method for detecting and associating allelic variants of SP-A2 gene with the disease and for prediction of an individual's predisposition to pulmonary tuberculosis.